Thin layer chromatography principle, application, case study

1. THIN LAYER CHROMATOGRAPHY
Presented by
Piyush M Chavan.
Guided by
Prof. K.B. Gabhane
VidyaBharati College Of Pharmacy, Amravati-444601
2019-2020
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2. INDEX
SR. NO TITLES
1 Introduction
2 Principle
3 Instrumentation
4 Working
5 Application
6 6.Case study
7 Result & Conclusion
8 Reference
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3. TLC is one of the simplest, fastest, easiest and least expensive of several
chromatographic techniques used in qualitative and quantitative analysis to
separate organic compounds and to test the purity of compounds
TLC is a form of liquid chromatography consisting of:
 A mobile phase (developing solvent) and
 A stationary phase (a plate or strip coated with a form of silica gel)
 Analysis is performed on a flat surface under atmospheric pressure and room
temperature
1.Introduction
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4. Definitions
 Thin Layer Chromatography can be defined as a method of separation or identification of
a mixture of components into individual components by using finely divided adsorbent
solid / (liquid) spread over a glass plate and liquid as a mobile phase.
 Synonyms: Drop, strip, spread layer, surface chromatography and open column
chromatography
- Adsorption or retention or partition or both or any other principle of a substance (s ) on
the stationary phase
- Separation of the adsorbed substances by the mobile phase
- Recovery of the separated substances by a continuous flow of the mobile phase (elution)
- Qualitative and quantitative analysis of the eluted substances
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5. Principle of TLC
It is based on the principle of adsorption chromatography or partition chromatography
or combination of both, depending on adsorbent, its treatment and nature of solvents
employed.
 The components with more affinity towards stationary phase travels slower.
 Components with less affinity towards stationary phase travels faster
 In TLC, a solid phase, the adsorbent, is coated onto a solid support (thin sheet of
glass,plastic, and aluminum ) as a thin layer (about 0.25 mm thick). In many cases, a
small amount of a binder such as plaster of Paris is mixed with the absorbent to
facilitate the coating.
2.Principle
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6.  The mixture (A + B) to be separated is dissolved in a solvent and the
resulting solution is spotted onto the thin layer plate near the bottom. A
solvent, or mixture of solvents, called the eluatant, is allowed to flow up
the plate by capillary action. At all times, the solid will adsorb a certain
fraction of each component of the mixture and the emainder will be in
solution. Any one molecule will spend part of the time sitting still on the
adsorbent with the remainder moving up the plate with the solvent. A
substance that is strongly adsorbed (say, A) will have a greater fraction
of its molecules adsorbed at any one time, and thus any one molecule of
A will spend more time sitting still and less time moving and vice versa.
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7. Separation of mixtures in microgram quantities by movement of a solvent across a flat
surface; components migrate at different rates due to differences in solubility, adsorption,
size or charge; elution is halted when or before the solvent front reaches the opposite side of
the surface and the components examined in situ or removed for further analysis.
Separations in TLC involve distributing a mixture of two or more substances between a
stationary phase and a mobile phase
1.The stationary phase: is a thin layer of adsorbent (usually silica gel or alumina) coated on a
plate.
2.The mobile phase: is a developing liquid which travels up the stationary phase, carrying
the samples with it. Components of the samples will separate on the stationary phase
according to:
how much they adsorb on the stationary phase versus?
how much they dissolve in the mobile phase?
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8. 1. STATIONARY PHASE
Adsorbents mixed with water or other solvents→ slurry
Silica gel H ( Silica gel with out binder )
Silica gel G ( Silica gel + CaSO4 )
Silica GF (Silica gel + binder + fluorescent indicator)
Alumina, Cellulose powder, Kieselguhr G( Diatomaceous
earth + binder
3.Instrumentation
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9. 9

10. 2. GLASS PLATE
Specific dimensions-
20cm Х 20cm, 20cm Х 10cm, 20cm Х 5cm
Microscopic slides can also be used
Plates should be of good quality & withstand high temperatures
3. PREPARATION & ACTIVATION OF TLC
PLATES
 Pouring ( simplest methods )
 Dipping (used for small plates )
 Spraying ( difficult to get uniform layers )
 Spreading ( best technique ) TLC Spreader
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13. 13

14. Activation of Plates
○ After spreading → Air dry (5 to 10 minutes)
○ Activated by heating at about 100˚C for 30 min.Then plates may be kept in
desiccators
4. APPLICATION OF SAMPLE
» Using capillary tube or micropipette
» Spotting area should not be immersed in the mobile phase
5. DEVELOPMENT TANK
▫ Better to develop in glass beakers, jars to avoid more wastage of solvents
▫ When standard method is used, use twin trough tanks
▫ Do chamber saturation to avoid “edge effect”
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15. 6. MOBILE PHASE
M.P used depends upon various factors
 Nature of the substance
 Nature of the S.P
 Mode of Chromatography
Separation to be achieved, Analytical/Preparative
e.g. → pyridine, carbon tetrachloride, acetone, water, glycerol, ethanol, benzene….
7. DEVELOPMENT TECHNIQUE
 One dimensional development
 Two dimensional development
 Horizontal development
 Multiple development
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16. 8. DETECTING OR VISUALISING AGENTS
>Non specific methods
•Iodine chamber method
•Sulphuric acid spray reagent
•UV chamber for fluorescent compounds
•Using fluorescent stationary phase
>Specific methods
Spray reagents or Detecting agents or Visualizing agents
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17. How to Run Thin Layer Chromatography?
• Step 1: Prepare the developing container
• Step 2: Prepare the TLC plate
• Step 3: Spot the TLC plate
• Step 4: Develop the plate
• Step 5: Visualize the spots
• Retardation factor:
4.Working
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18. 18

19. Factors affecting Rf value
It depends on following factors:
Nature adsorbent
Mobile phase
Activity
Thickness of layer
Temperature
Equilibrium
Loading
Dipping zone
Chromatographic techniques
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20. »Purity of sample
»Examination of reaction
»Identification of compounds
»Biochemical analysis
»In pharmaceutical industry
»Separation of multicomponent pharmaceutical formulations
»In food and cosmetic industry
5. Application
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21.  Detection Of Ranitidine By Thin Layer Chromatography Technique-A Case Study
6. Case study
Abstract
In one case, viscera of a lady was received, who expired following injection given
by doctor in urban area. After conducting elaborate analysis data were found
similar to Ranitidine. Hence, it was thought worthwhile to concentrate on this
drug .The present paper describes the extraction of Ranitidine from visceral
material and its identification by thin layer chromatography using suitable solvent
system and potassium iodo bismuthate as locating reagent
Fig :- Structure of Ranitidine 21

22.  Extraction of Ranitidine from autopsy material:
In a portion of about 100 g each of various biological tissues (stomach, intestine,
liver spleen and kidney) containing the g above mentioned drug, 10g ammonium
sulphate was added and minced in an aqueous solution. Then biological sample was
made alkaline with the help of ammonia and sample was extracted in a separating
funnel with 150 ml of mixture of ethyl alcohol: water (10:90) was added this was
again extracted with 100 ml mixture of ethyl alcohol: water (10:90) The filtrate were
combined and evaporated up to 10 ml. The extracts were combined and subjected
to clean up by passing through the mixture of silica gel g and activated charcoal
filled column having glass wool at the bottom. Finally in the collected filtrate was
evaporated up to 1 ml over hot water bath and can directly used for identification
Ranitidine. 22

23.  Thin layer chromatographic analysis
 A standard glass TLC plates was coated with slurry of silica gel G in water to a
uniform thickness of 0.25 mm. The plate was activated by heating in an oven at
110oC for about one hour. Aliquots of standard Ranitidine and extract obtained
from autopsy material were spotted on to the plate, which was developed with
Methanol: Ammonia (100:1.5) in a pre saturated TLC chamber, to a height of 10
cm. The plate was removed from the chamber dried in air and sprayed by
Potassium iodo Bismuthate reagent that gave orange colored spot at 0.53. The Rf
value of Ranitidine can be compared with the obtained spots of visceral extract.
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24.  The prescribed developed method and the analysis of Ranitidine in visceral
tissue could prove that the injection Ranitidine was given to the lady. This
method is sensitive and can detect ranitidine up to 10µg concentration by thin
layer chromatography and can be used for routine analysis. The question before
investigating officer in any death alleged to have occurred due to drug reaction,
is “Whether deceased was given Ranitidine (or other drug) injection ?”. By such
meticulous analysis it can be opined in a positive manner and hence sufficient to
establish that the alleged drug had been given. However, the subsequent
questions of drug over dose, adverse reaction and its contribution to death
need considerations like circumstantial evidence, post mortem findings etc.
7.Results And Discussions
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25. 1. Indian Pharmacopoeia Vol. I 1996 published by j the controller of publication Civil
Lines, Delhi -54.
2. Ettinger, M.B., Ruchhof, E.C. and Lisha, R.J. Anal. Chem. (1951), 23, 1783.
3. Sharma H L and Sharma KK. Principles of Pharmacology, Paras Publishing,
Hyderabad ( 2007),p.394.
4. Stahl, E.L. (1969) “Thin layer chromatography A Laboratory hand book”,
Springer international student edition, New York
5. E.G.C. Clarke, Clarke isolation and identification of drugs in pharmaceuticals,
body fluids and Post-mortem material second edition, The Pharmaceutical Press,
London (1969).
8.References
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